1. Field of the Invention
This invention relates to a novel method and apparatus for encapsulating discrete droplets of liquid by generating a continuous coating or layer of a polymerizable liquid which is substantially immiscible with the core liquid.
2. Background
Encapsulation refers to processes whereby an active ingredient is placed into a stabilized form in order to allow it to be conveniently stored, or protected from unfavorable conditions, until needed. The active ingredient may be dispersed in a protective matrix, or it may be surrounded by a coating, a shell, or a membrane. The release of active ingredient from the protected form may be rapid (such as by crushing, or by ingestion), or gradual (such as by dissolution, diffusion, or bio-degradation). In this manner it is possible to maximize the effectiveness of the active ingredient by ensuring that it is released at the proper time. This xe2x80x9ccontrolled releasexe2x80x9d can also be made to occur over a programed time interval (sustained release), or on demand (stimulated release).
The term xe2x80x9cmicrocapsulexe2x80x9d has been used to describe small particles or beads, which range in size from less that one micron, up to several millimeters, which may contain a wide variety of active ingredients (Thies, 1994; Thies, 1987; Goodwin, 1974; Deasy, 1984; Hegenbart, 1993). Microcapsules can be divided into two broad groups: (1) xe2x80x9cAggregatexe2x80x9d type microcapsules have the active ingredient dispersed uniformly throughout a continuous matrix. The matrix may be a solid dry polymer or a gel swollen with solvent. In the case where the gel is swollen with water, the term xe2x80x9chydrogelxe2x80x9d is applied. Hydrogel encapsulation systems of this type are generally based on cross-linked forms of water-soluble polymers such as alginate, gelatin, pectin, agar, gellan, or starch (Sanderson, 1989). (2) xe2x80x9cMononuclearxe2x80x9d microcapsules, on the other hand, consist of materials which show a true xe2x80x9cshell-corexe2x80x9d morphology. These are similar to an egg in that they have a solid shell or flexible membrane surrounding a core which may be a liquid, a solid, or even a gel.
Methods of producing microcapsules are the subject of several review articles (Sparks, 1981; Benita; 1996, Thies, 1994; Goodwin, 1974; Deasy, 1984; Hegenbart, 1993). Although numerous methods are described in these articles, the majority are simply not suitable for producing large ( greater than 500 micron diameter) mononuclear microcapsules which show a true shell-core morphology, and are capable of containing an aqueous-based solution as a core. Such capsules can be prepared with some degree of success, however, by using a method termed xe2x80x9cconcentric extrusionxe2x80x9d. In this approach to microcapsule manufacture, two mutually immiscible liquids are extruded through concentric orifices in order to produce a biliquid column, with the core fluid on the inside. Under the influence of gravitational or other forces, this biliquid column fragments into discrete droplets having a shell/core morphology. The liquid shell is then made to harden by some mechanism to give liquid-core microcapsules with a solid shell.
Hardening of the shell is generally effected either by heating to remove a solvent, or by cooling to solidify the molten shell material. The outer coating in these systems is often either a molten wax, or a solution of aqueous polymer such as gelatin or alginate. The use of heat, either to melt the shell material, or to drive off solvent, can be detrimental to sensitive core materials such as protein solutions or suspensions of living organisms. Similarly, the use of solvent-based shell formulations can lead to undesirable contamination of the core material, as well as health and safety concerns. Aqueous-based shell formulations such as gelatin cannot be used in conjunction with aqueous core materials since phase incompatibility is a necessary prerequisite for formation of a shell/core morphology using this technique. Also, these types of shells are, by nature, easily affected by water, and also very susceptible to dehydration. Another drawback of the existing techniques is that the physical and mechanical properties of the shell materials suitable for use in these approaches are limited. Waxes, for instance, have very poor elasticity and mechanical strength, and also low melt viscosity which makes production of very thin membranes impractical. Low molecular weight thermoplastic polymers are generally too brittle and lack the flexibility to give strong, thin-walled, individual capsules. In fact, very few polymeric shell materials have melting points low enough to make existing approaches widely practical. Thin, flexible, and durable membranes are generally only associated with crosslinked elastomeric polymers. By nature, such polymers are insoluble and will not melt even at extreme temperatures, so they cannot be used in liquid form. It has been demonstrated that even though some strong high molecular weight thermoplastic polymers have suitably low melting points, they tend to xe2x80x9cfiberizexe2x80x9d rather than give individual droplets when extruded through an orifice. Related microcapsule fabrication techniques such as xe2x80x9ccentrifugal extrusionxe2x80x9d suffer from similar drawbacks.
Examples of these existing techniques and their shortcomings can be found in various U.S. patents. Probably the first use of concentric fluid streams to accomplish the encapsulation of liquid agents was described in U.S. Pat. No. 2,275,154 (Merrill et al., Mar. 3, 1942). In that invention a medicinal component is surrounded by gelatin in a liquid form, and then the gelatin is caused to harden. Since gelatin is soluble in water, this method is useless for the encapsulation of aqueous liquids.
Similar methods are reported in the following U.S. Pat. No. 2,766,478 (Raley et al., Oct. 16, 1956); U.S. Pat. No. 2,799,897 (Jansen et al., Jul. 23, 1957); U.S. Pat. No. 2,911,672 (van Erven Dorens, Nov. 10, 1959); U.S. Pat. No. 3,015,128 (Somerville, Jan. 2, 1962); U.S. Pat. No. 3,310,612 (Somerville, Mar. 21, 1967); U.S. Pat. No. 3,389,194 (Somerville, Jun. 18, 1968); U.S. Pat. No. 3,423,489 (Arens et al., Jan. 21, 1969); and U.S. Pat. No. 3,779,942 (Bolles, Dec. 18, 1973). The shortcomings of these methods are described above, and are also discussed in U.S. Pat. No. 3,423,489. More recently, these shortcomings have been discussed in U.S. Pat. No. 5,478,508 (Suzuki et al., Dec. 26, 1995). This patent fails to successfully overcome the stated shortcomings, as it utilizes materials such as waxes, oils, fats, paraffins, thermoplastic resins, gelatin, or other water-soluble polymers for the shell material. The insufficiencies of these coatings have been described above.
Currently, there is a small specialty market for the sale of beneficial insects (such as lady beetles and parasitic wasps) for use in pest control on high value crops, such as greenhouses and nurseries producing xe2x80x9corganically-grownxe2x80x9d produce. Since the cost of naturally-produced beneficial insects is high, the resulting xe2x80x9cgreenxe2x80x9d produce that goes to market is sold only to a small number of customers willing to pay substantially higher prices. While mass rearing of phytophagous insects (plant feeding insects) is well developed and has been implemented in most entomological research organizations world-wide, mass rearing of entomophagous insects (insects that eat other insects) generally lags far behind. The reason for this large gap in rearing methods has been largely due to the lack of a suitable artificial substitute for the natural insect host diet. Although useful liquid diet formulations are currently being developed, there exist no suitable technologies for incorporating these aqueous-based liquid diets into practical forms for storage and presentation. Liquid-core hydrocapsules are a promising solution to this problem.
A laboratory method for encapsulating artificial diet for rearing predators of harmful insects by forming a paraffin coating over an artificial liquid diet has been described by Hagen and Tassan (1965). This method utilized a molten waxy polymer for the coating material, and as such it has limited utility. Also, only a few hundred coated droplets could be produced per day, as the method was very labor intensive, and thus not practical for large-scale mass rearing. An improvement over the Hagen and Tassan method was developed by Cohen (1983) for producing paraffin coated artificial diet capsules. This method lends itself to improved uniformity of the droplet size and efficiency in formation. The main drawback, however, is that the method required some care and patience on the part of the technician, who could produce no more than 2-3,000 capsules per day. Thus, the technique is not suitable for mass production rearing, but simply useful as a research tool.
The closest commercial application to what is needed for production of individual artificial diet capsules that are suitable for use in general beneficial insect rearing is a process which produces 1-2 mm diameter wax-based coated artificial diet for Chrysoperla carnea (green lacewing) predators. This method is based on earlier procedures developed by Martin et al. (Martin, 1978). This encapsulated diet is not suitable for general mass rearing for several reasons: The diet coating melts at temperatures above 28xc2x0 C., which is slightly above indoor room temperature, and would be unusable in warm incubators, greenhouses, or outdoors in even the mildest of growing regions. The size of the capsules produced are too small to contain sufficient diet for optimal rearing of many predators and parasitoids. The wax coating cannot withstand rough handling nor packing in large containers, which could cause these tiny wax capsules to crush or leak. The biggest limitation to the wax coating used, is that it cannot contain any insect diet with a high lipid (fat) content, such as the USDA""s DI-DIET (see U.S. Pat. No. 5,799,607, Sep. 1, 1998, Greany et al.). The lipids act as a solvent which decomposes the wax coating causing the capsule to leak. Currently, an ideal encapsulation system is not available. Such a system would need to meet the following criteria: be acceptable for feeding by the insects of concern; be penetrable by the feeding insect; not be deleterious to the feeding insect; not interact negatively with, or alter the properties of the artificial diet; maintain integrity at ambient temperature; contain an aqueous solution without dissolving; prevent desiccation of the liquid diet; allow itself to be formed or applied in various thicknesses; permit any 3-D geometric shape, generally spherical in nature; be suitable for various sizes, ca.1.0 cmxc2x10.5 cm; withstand sterilization by irradiation (or other means), which is necessary for long term storage.
There are no existing methods applicable for the mass-production of soft-shelled, water-filled, 1 to 10 mm diameter capsules which can be used to encapsulate aqueous artificial diets (or any water-based solution). Industrial encapsulation technologies in use today (developed for the needs of the biomedical, pharmaceutical, and food industries), generally produce microcapsules that are too small (xe2x89xa650 xcexcm), or too hard, or are made of materials that are unsuitable for a wide variety of applications. Many of these methods rely on water to dissolve the capsule material in order to release its contents, (such as in the human stomach). Obviously, such methods are not well suited for containing aqueous-based liquids. U.S. Pat. No. 4,096,944 (Simpson, Jun. 27, 1978) describes a method which utilizes microcapsules having diameters in a range of 400 to 5000 microns, with inert, frangible shells enclosing droplets of liquid water. These microcapsules may have some suitability for the applications described therein; however they are made in accordance with the method disclosed in U.S. Pat. No. 3,389,194 (discussed above), and as such they are not broadly useful for the aforementioned reasons.
One proven approach for chemical-alternative insecticides is the use of insect pathogens or xe2x80x9centomopathogensxe2x80x9d as a bio-rational method of controlling pests. Entomopathogens are naturally-occurring disease-causing organisms such as protozoa, bacteria, fungi, and nematodes which specifically infect or vector other harmful agents (such as endotoxins) into insects causing death or disruption of its life cycle. They are very good candidates for use as bio-pesticides since most insect pathogens are specific to certain groups of insects or certain life stages of insects. Additionally, microbial entomopathogens generally do not directly affect beneficial insects and are non-toxic to wildlife or humans (Weeden et al., 1996; Hoffmann and Frodsham, 1993). Entomopathogens generally infect their host (pest) insects through ingestion or by direct contact with an organism. In either case, once the pathogen has entered the insect, it will eventually lead to the insect""s death or decreased activity of the pest. Widespread use of this type of pest control strategy has been stifled greatly by the lack of suitable methods for delivery of entomopathogens into the environment. Most entomopathogenic organisms are extremely sensitive to adverse environmental conditions. Packaging of microorganisms in the form of microcapsules can provide extended useful lifetimes by protecting them from harsh conditions such as sunlight and low humidity.
The same capsules which are useful in the mass-rearing of beneficial insects can be converted into lethal snacks for pest insects, simply by incorporation of entomopathogenic agents into the aqueous core. For applications such as cockroach and fire ant baits, it is not necessary that the shell be soft enough to allow the entrapped organisms to escape on their own, since the shells can be easily breached by the feeding insect. In fact, the capsule can serve as a convenient package which allows the target insect to carry the infective agent directly into its nest. This behavior has been observed in studies involving wild fire ants feeding on encapsulated artificial diet prepared using the method of the current invention. Incentive to feed on the capsules can also be provided by incorporation of essential nutrients, or by the addition of feeding stimulants or kairomones. For more passive delivery approaches, it is possible to adjust the shell formulation to the needs of a particular system, so that the entomopathogenic agent can emerge without direct contact between the insect and the microcapsule. In fact, a combination of the two release mechanisms can serve to be the most effective approach in some cases. Other uses for microencapsulated microorganisms are possible as well. Some of these applications would include: fermentation processes, herbicides, medical, veterinary, and horticultural uses.
There are several reports in the literature concerning the microencapsulation of bio-control microorganisms in continuous-matrix hydrogels. These processes do not result in actual shell-core microcapsules as discussed above. Instead, they result in soft, hydrated gel particles which are essentially a uniform distribution of active organisms trapped in a hydrogel matrix, rather than a contained liquid suspension of organisms. These hydrogels have a very high water content (up to 90%), and thus they are very susceptible to dessication. They also serve as prime breeding grounds for non-desirable contamination by ambient microorganisms. This type of hydrogel-entrapment method has been used for the encapsulation biocontrol fungi such as Trichoderma, Gliocladium, Alternaria, and Penicillium into alginate-clay matrices for use as mycoherbicides (Walker, 1983; Fravel, 1985). The alginate hydrogel particles were then dried to produce hard pellets. This delivery system was found to be somewhat effective, but subsequent bacterial contamination of the pellets was apparent. Such contamination is no doubt facilitated by the porous and hygroscopic nature of the entrapping polymer. A true shell-core type of microcapsule having an aqueous interior surrounded by a protective membrane, rather than simply a gel matrix would help to prevent this.
A substantially similar method for encapsulating Steinernematid and Heterohabditid nematodes has been reported (Kaya, 1985; 1987). This work serves as the basis for the following U.S. Pat. No. 4,615,883 (Nelsen et al., Oct. 7, 1986); U.S. Pat. No. 4,701,326 (Nelsen et al., Oct. 20, 1987); and U.S. Pat. No. 4,753,799 (Nelsen et al., Jun. 28, 1988). The survival rate and pathogenicity of the nematodes remained high throughout the encapsulation process, but dessication during storage proved to be a problem. Attempts have been made to reduce the rate of evaporation of the aqueous carrier by coating these continuous-matrix capsules with hydrocarbon oils or greases, as described in U.S. Pat. No. 4,178,366 (Bedding, Dec. 11, 1979) and U.S. Pat. No. 5,401,506 (Chang et al., Mar. 28, 1995).
A method of encapsulating cells in a tubular extrudate is described in U.S. Pat. No. 5,389,535 (Aebischer, et al., Feb. 14, 1995). The cells are encapsulated within a semi-permeable polymeric membrane by co-extruding an aqueous cell suspension and a polymer solution through a common port to form a tubular extrudate having a polymeric outer coating. The cell suspension and the polymeric solution can be extruded through a common extrusion port having at least two concentric bores, such that the cell suspension is extruded through the inner bore and the polymeric solution is extruded through the outer bore. The polymeric solution coagulates to form an outer coating. As the outer coating is formed, the ends of the tubular extrudate can be sealed to form a cell capsule. In one embodiment, the tubular extrudate is sealed at intervals to define separate cell compartments connected by polymeric links. This method is not suitable for the production of discrete microcapsules. Also, the semipermeable membranes produced by this method are not suitable for long term storage without dessication.
U.S. Pat. No. 5,364,634 (Lew, Nov. 15, 1994) described a controlled-release pH-sensitive capsule. The microcapsules are made by methods well known in the art (such as centrifugal extrusion), and as such are subject to the limitations described above. Similar limitations are encountered in the method of U.S. Pat. No. 4,888,140 (Schlameus et al., Dec. 19, 1989) which describes forming fluid filled microcapsules by the simultaneous extrusion of core and shell material from coaxially aligned and concentric extrusion nozzles into a surrounding carrier fluid. In this method an aqueous polymer such as gelatin is used as the shell material. The limitations of such materials have been described above. U.S. Pat. No. 4,948,586 (Bohm et al., Aug. 14, 1990) describes a method for producing microencapsulated insecticidal pathogens. This method is based upon emulsion formation, and the product is described by the inventor as xe2x80x9canalogous to gelatin ballsxe2x80x9d. It does not yield discrete microcapsules having a shell/core morphology and a liquid center.
The current invention allows one to produce capsules that are of the shellcore type, and consist of a polymer membrane surrounding a liquid center. An important feature of these mononuclear microcapsules is that they contain a water-based core. Other types of processes, such as the familiar xe2x80x9csoftgelxe2x80x9d technology used to encapsulate vitamin E are not suitable for encapsulating aqueous liquids (Rose, 1987). These types of methods are described, for instance, in U.S. Pat. No. 4,744,988 (Brox, Dec. 19, 1989). The shell materials resulting from the unique encapsulation process described in the current patent disclosure are crosslinked hydrophobic elastomeric polymer networks. These shells are produced via the ultraviolet (UV)-initiated free-radical copolymerization of functionalized prepolymers (silicones, urethanes, epoxys, polyesters, etc.) and/or vinyl monomers such as acrylates. Because the structure of the polymer shell of these types of capsules is very distinct from the softgels or aggregate-type hydrogel microcapsules described above, they are referred to as xe2x80x9cHYDROCAPSULES(trademark)xe2x80x9d. This implies that they have an aqueous liquid core surrounded by a thin hydrophobic polymer membrane. However, further research has demonstrated that this encapsulation process also has utility for encapsulating liquids other than xe2x80x9caqueous compositionsxe2x80x9d. The same method used for encapsulating xe2x80x9caqueous compositionsxe2x80x9d has been utilized to encapsulate oils and alcohols. These xe2x80x9cnon aqueous compositionsxe2x80x9d, are able to be encapsulated because they too are substantially immiscible with the shell-forming polymerizable liquid.
This encapsulation process has other potential applications in the agricultural community, and in the biomedical and chemical industries. The current invention enables the production of small liquid-containing polymer capsules, that have good chemical resistance to many organic solvents, and are capable of containing many types of active ingredients for use in a wide range of applications including (but not limited to): food sources for other insects used in laboratory research; poison baits for pest insects (such as roaches or fire ants) which could safely contain toxins combined with a phagostimulant or other attractant (xe2x80x9cattracticidexe2x80x9d approach); pheromone release for insect mating disruption; controlled pheromone release for traps used in insect population monitoring; water-soluble drug, medicine, or microbial dispensing systems; or for encapsulation of foodstuffs or flavoring ingredients, and similar applications.
A method is disclosed for encapsulating discrete droplets of liquid by generating a continuous coating or layer of a polymerizable liquid which is substantially immiscible with the core liquid. The polymerizable liquid is made to surround discrete droplets of the core liquid and is made to polymerize to form a shell, membrane, or solid coating around the discrete droplet core liquid. Methods of making and using the encapsulated liquid as well as an apparatus for making the hydrocapsules are also disclosed.